Cartridge, system and method for automated medical diagnostics

ABSTRACT

Cartridge for the detection of the presence, absence and/or amount of a target nucleotide sequence in a sample. The cartridge includes one or more nucleic acid sequences, wherein the cartridge has a generic part and one or more separate application-specific parts, which are connectable to the generic part.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 11/917,955 which is a U.S. National Stage of InternationalPatent Application No. PCT/IB2006/051941 filed Jun. 16, 2006 whichpublished as WO/2006/136990 on Dec. 28, 2006, and claims priority ofEuropean Application No. 05105608.3 filed Jun. 23, 2005. The disclosuresof International Patent Application No. PCT/IB2006/051941 and U.S.patent application Ser. No. 11/917,955 are expressly incorporated hereinby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention pertains to a cartridge for the detection of the presence,absence or amount of specific DNA or RNA sequences. The invention alsopertains to the use of a system, optionally incorporating a cartridge,for the detection of the presence, absence or amount of specific DNA orRNA sequences.

2. Discussion of Background Information

Since the discovery of DNA, the technology relating to the detection ofthe presence, absence or amount of specific DNA or RNA sequences in asample has taken an enormous flight. Especially PCR, the PolymeraseChain Reaction has contributed enormously to the development of assaysof all types for the detection of the presence or absence of DNA or RNAsequences. At present, it is possible to collect DNA containing samplesfrom an organism and determine the presence, absence or amount thereinof certain specific DNA sequences (target sequences). Technology isavailable to perform such analysis for multiple target sequences at thesame time, so-called multiplex detection of target sequences to therebyincrease throughput.

At present, this type of analysis is not yet performed on a routinebasis, such as for instance the measurement of the blood-glucose contentin the case of diabetes. Generally, well-equipped laboratories arenecessary, and careful protocols have to be used in order to avoidcross-contamination and to ensure that the results obtained are reliablei.e. false-positive or false-negative readings of the tests areminimized. However, as still a lot of manual labor is involved ofextensively trained and supervised personnel, there remains a need inthe art to overcome the above disadvantages of the present methods ofDNA or RNA analysis. Especially RNA analysis is known to be verydifficult because contamination happens very easy due to the present ofminute amounts of RNA in the atmosphere and on the hands of the skilledanalysts. Furthermore, the present methods of analysis are not onlylaborious, they are also time-consuming. Typically, an efficientprocedure for a conventional DNA or RNA analysis takes about 6 hours dueto, inter alia, all the handling between the various systems for thetaking of samples, the isolation of DNA or RNA from the sample, thesubsequent assay for the analysis of the presence, absence or amount ofthe target sequence in the sample, the processing of any resultsobtained and the corresponding presentation of the results.

Cartridge-based systems for the detection of DNA have been disclosedbefore.

For example U.S. Pat. No. 5,882,903 discloses a system for the detectionof DNA. The system comprises a first assembly having one or morereaction chambers and a second assembly comprising a number of fluidchambers. The fluid chambers each hold fluid which is used during thedetection of the DNA. These fluids comprise washing fluids, lysis fluid,and an amplification solution containing an amplification buffer andappropriate primers. The reaction chambers are used to perform thedifferent steps of the detection such as washing, lysis, andamplification.

Other cartridge based systems known from prior art and used for thedetection of DNA are for example disclosed in U.S. Pat. No. 5,585,069,U.S. Pat. No. 6,168,948 and WO97/27324.

A disadvantage of the known cartridge-based system is that thecartridges of these systems are designed as a single body. The knowncartridge does not offer any flexibility towards the application forwhich the cartridge is intended to be used, for example for specificbacteria to be detected with the system or for different kinds ofsamples to be introduced in the cartridge.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a cartridge for thedetection of the presence, absence and/or amount of a target nucleotidesequence in a sample, which provides for a more flexible use of suchcartridge.

By providing a generic part which can be used for a wide variety ofapplications and one or more application-specific parts which arespecifically configured to be used for a certain application, thecartridge can be assembled on the basis of the specific application forwhich it is intended to be used.

The generic part may typically comprise fluid handling means such aspumps and valves, a number of process chambers which are usedindependently of the application of the cartridge, and fluid storage andwaste rooms for different fluids, such as lysis and washing buffers.These elements of the cartridge will hereinafter be described in moredetail.

In an embodiment one of the one or more specific parts is a PCR bodyhaving one or more thermocycling chambers and comprising a number ofprimers. It may be possible that the cartridge is to be used fordifferent panels of bacteria/resistances. By providing different PCRbodies comprising a number of primers, an application-specific PCR bodycan be chosen on the basis of the application for which the cartridge isused. For instance the PCR body comprising the primers can be selectedon the basis of the panels of bacteria/resistances that are to bedetected, which selection may be specific for a particular assay or fora particular region, such as Europe, Asia or Africa. It is also possiblethat the size or the number of thermocycling chambers is selected on thebasis of the bacteria/resistances to be detected. In a preferredembodiment the thermocycling chambers are only used for thethermocycling step. The process chambers, in particular the primers, arein such embodiment not affected by any other step performed in thecartridge.

In an embodiment there are provided two or more thermocycling chambers.In each of the thermocycling chambers an amount of process fluid can bebrought, which makes an efficient parallel processing possible.

In an embodiment a primer is arranged in each one of the one or morethermocycling chambers. By arranging the primers in the one or morethermocycling chambers the primers do not have to be transferred beforethe thermocycling steps can be performed. In this way a more efficientuse is made of the primers. Furthermore, this allows a more simpledesign of the PCR body.

In an embodiment the primer is spotted on the PCR body. Such spottingmay be done with any known spotting technique, such as for instance inkjet printing. Preferably, the spotted primers are provided in the one ormore thermocycling chambers, but they may also be provided in any othersuitable location such as the inlet channels for the sample leading tothe one or more thermocycling chambers.

In an embodiment one of the one or more application-specific parts is adetection device. A number of different detection methods can be used todetect the amplicons in the sample after the DNA/RNA amplification. Suchdetection methods may include optical, electrochemical, magneticcapillary and gel-electrophoresis detection methods. Depending on thedetection method to be used for a certain sample the detection device,or at least a part thereof may be selected and connected to the genericpart of the cartridge.

In an embodiment the detection device is selected on the basis of thePCR body that is used for the detection of certain bacteria/resistances.Certain detection methods are typically related to the specificbacteria/resistances which are to be detected, and therewith also withthe primers which are used in the detection method. As a result the PCRbody with primers and the detection device may be selected as a pair,i.e. with the section of a PCR body also the selection of the detectiondevice is made.

It may be possible that only a particular part of the detection device,such as a substrate and/or substrate holder may be specific for thedetection of a certain DNA/RNA. It is therefore contemplated that theapplication-specific detection device may only be a part of the actualdetection system used in the cartridge to make the detection of aparticular DNA/RNA possible.

In an embodiment of the invention one of the one or moreapplication-specific parts is a sample introduction device. Dependent onthe quantity of the sample needed, the type of sample, the state inwhich it is provided, different sample introduction devices may be usedfor introduction of a sample into the cartridge. The use of sampleintroduction device further provides an easy and secure connection tothe cartridge, and there with an easy and reliable introduction of thesample in the cartridge.

In an embodiment the sample introduction device is a pre-lysis deviceconfigured to prepare a sample to a specific state. The generic part ofthe cartridge is designed to process a sample in a particular samplestate, for instance fluid. A pre-lysis device may be provided to processthe sample from a particular state in which it is available, and inwhich it cannot be used in the cartridge, to the state for which thecartridge is designed to process it. Such state may be to which is driedout fluid, fluid being present in a solid carrier, and such. The processin the pre-lysis may be performed before the pre-lysis device isconnected to the generic part, but also after that this connection ismade.

In an embodiment at least one of the one or more of theapplication-specific parts is provided with an identification device. Toavoid errors in the selection of the application specific parts it isuseful to provide an identification on the application-specific parts sothat easily can be checked whether the correct application-specific partor set of parts is combined with the generic part.

Such identification device may include stickers, bar codes, color codes,magnetic codes and such. Preferably, an automatic identification system,such as a RF-tag identification system, is used. Such more advancedidentification devices may also be used to track the location history ofthe generic part and/or the application specific parts. It may forinstance be important that a certain application-specific part iscooled. With the use of a location history track system it can bechecked whether the application-specific part has not been too long outof a cooling device. In a preferred embodiment a control unit of thedetection system checks whether the correct application-specific partshave been connected with the general part and whether the generic partand application specific parts still fulfill all requirements regardinglocation history and such.

The present invention typically avoids or minimizes manual labor, avoidscross contamination, provides faster results that are more reliable, isuser friendly and is easily adapted for the analysis of different targetsequences. The present invention provides for a high throughput methodfor the analysis for the presence, absence or amount of DNA or RNA inany type of sample, preferably blood.

The present invention provides for a cartridge that is suitable for thedetection of the presence, absence or amount of DNA and/or RNA. Thedetection of the presence, absence or amount of DNA and/or RNA isindicative, for instance, for the presence, absence or amount of a gene,an allele of a gene, a genetic trait or disorder, a polymorphism, asingle nucleotide polymorphism (SNP) or of the presence of exogenous DNAor RNA in an organism, i.e. the presence, absence or amount of pathogensor bacteria in organisms.

Through the present invention, suitable remedies can be developed forthe preparation of medicaments for the treatment of the so diagnosedailment. For instance, the detection in a sample (say, blood) from anorganism (say, a human) of a pathogen (say, a virus) may thus lead tothe diagnosis and the corresponding treatment (say, an antibiotic).

The cartridge may be of an exchangeable type which can be positioned ina reusable apparatus. Such cartridge may be disposable, recyclable orreusable, possibly after cleaning. By providing an exchangeablecartridge all parts that may come into contact with the sample may afterthe detection process be taken out of the apparatus and the cartridgemay be exchanged for another one or cleaned before a next use. In otherembodiments the cartridge may be an integral part of the reusableapparatus which is cleaned after each use.

In certain embodiments the apparatus comprises a control unit forcontrolling the isolation means, amplification means and/or thedetection means. The control unit makes an automatic control of theisolation of DNA, the amplification of DNA and the detection of theamplified DNA possible.

The cartridge comprises one or more chambers in which the sample is heldduring the detection process. Such chambers may comprise an introductionchamber for introducing a sample in the cartridge, a lysis chamber forlysis of the cells in the sample, a washing chamber for washing, one ormore thermocycling chambers for the amplification of the DNA, and adetection chamber which makes detection possible. It is also possible toprovide a single chamber for one or more of the functions described inrelation to the chambers. In such embodiment two or more chambers of theintroduction chamber, lysis chamber, washing chamber, the thermocyclingchamber(s), and the detection chamber may be combined in one singlechamber.

During the different steps of the detection process the sample will bein a respective chamber. For this purpose, the sample will betransferred from one chamber to another chamber between two processsteps. To make such transfer possible, each chamber is at leastconnected with another chamber by a fluid channel. In at least one, butpreferably each of these fluid channels a valve means may be provided,which valve means preferably normally closes the fluid channel, butopens the fluid channel upon actuation of the valve means therewithplacing the respective two chambers in fluid communication. The valvemeans may be designed as a one-way valve.

In certain embodiments the valve means are actuated by a valve actuationdevice. This valve actuation device is preferably arranged in thereusable apparatus.

In certain embodiments pump means are provided to pump the sample or anyother fluid used in the detection process such as lysis buffer,reagents, washing and separation buffers, pre-amplification buffers,from one chamber to another chamber. These pump means may be actuated bypump actuation means which are preferably arranged in the reusableapparatus.

In certain embodiments, the system comprises means for data collectionand/or means for data processing. These means are intended for use inthe analysis of the detected DNA and/or for the interpretation of theresults. In particular, in certain embodiments the data processing meansthat are able to link the presence, absence or amount of the targetnucleic acid (or combination thereof) to a particular diagnosis. Such adata processing means can for example be in the form of a computer incombination with a database.

In certain embodiments, the system can also comprise the means for theintroduction of one or more samples. Such sample introduction means maycomprise any suitable device, such as a holding or docking device forthe introduction of a sample from a syringe or pipette or such and mayfor instance comprise a one-way inlet valve, a septum, filters, and anoverflow.

In certain embodiments, the system can also comprise lysis device. Inthe lysis device, that can be under the control of a control unit, thesample is treated to provide any nucleic acids in the sample in a formthat they can be isolated from the sample. This lysis step typicallyincludes the lysis of the cells such that cell and/or nuclear membranesare ruptured to thereby free the nucleic acids contained therein. Usecan be made of means of physical or mechanical manipulation for thelysis step, but also chemical means can be used for lysis of the cellsin the sample, such as a lysis buffer. Means for mixing can be providedto mix the sample and the lysis buffer Methods for lysis of cells arewell known in the art from textbooks etc. If necessary such methods canbe adapted for use in the present system. Any waste that is produced bythe lysis step can be discarded, for instance to a waste device.

In certain embodiments, the sample insertion device and the lysis devicecan be combined.

In certain embodiments, the system can also comprise an enrichmentdevice, optionally under the control of a control unit. The enrichmentdevice enables the isolation of DNA from the lysed sample. To this endthe enrichment device may be equipped with means for the isolation ofDNA, such as magnetic particles. In this embodiment, the DNA or RNA ofthe present invention is absorbed onto magnetic particles. The absorbednucleic acid material can be subjected to one or more washing, drainingand/or purifying steps to remove any unwanted material such as remainsof biological material contained in the sample and other samplecomponents that are not DNA and/or RNA. When the absorbed DNA or RNA isof a desired purity, it can be desorbed or eluted from the magneticparticles. The enrichment device can also be equipped with means forphysical or mechanical manipulation of the fluids for mixing, separatingand isolating the DNA or RNA.

In certain embodiments, the system can also comprise the reagents thatare necessary for the enrichment step, i.e. the isolation of the DNA orRNA, such as buffers, washing fluids, water, filters, magnetic beadsetc.

In certain embodiments, the system can also comprise a waste device toaccommodate any waste produced from the enrichment step such as usedbuffers, washing fluids and the like.

In certain embodiments, the different waste devices of the system can beseparate for each different purpose or volume. In certain embodiments,two or more of the waste devices described herein can be combined toaccommodate all waste that is produced by the method of the presentinvention.

In certain embodiments, the system further comprises a pre-amplificationdevice, optionally under the control of a control unit. Thepre-amplification device can be used, for instance for increasing thetotal amount of DNA or RNA to be analyzed. Subjecting DNA or RNAobtained from the isolation step to a pre-amplification step canincrease the total amount of DNA. This is advantageously, especially inthe case of multiplex analysis, where multiple tests are performed onthe isolated DNA, for instance to detect the presence absence or amountof multiple pathogens in one sample at a time. Suitable technology isavailable in the art for increasing the amount of DNA and is generallyknown as Whole Genome Amplification.

In the pre-amplification device, the isolated and purified DNA or RNAcan be pre-treated with, inter alia an pre-amplification buffer and incase of whole genome amplification, with enzymes and DNTPs. Thepre-amplification device can be connected to a waste device for thedisposal of materials.

In certain embodiments, the pre-amplification device can also be usedfor carrying out certain assays for the detection of specific nucleicacids. Examples thereof are OLA-PCR like technologies such as providedby Applera (SNPplex), Keygene (SNPWave) and MRC-Holland (MPLA).

In certain embodiments, the system comprises an amplification device.The amplification device can be under the control of a control unit. Theisolated DNA, optionally pre-treated as described herein elsewhere, issubjected in the amplification device to an amplification treatment inthe amplification device. The amplification treatment comprises bringingthe isolated DNA in contact with a set of PCR primers that are specificfor the target nucleic acid, PCR enzymes such as one or more polymerasesand dNTPs.

In certain embodiments, the amplification device holds a plurality ofchambers. The plurality of chambers enables the isolated orpre-amplified DNA or RNA to be divided in portions and distributedamongst the chambers. In each chamber, an amplification step can beperformed using a different set of primers. In this manner, multiplexanalysis is provided in that one sample can be analyzed for thepresence, absence or amount of different target nucleic acids. IN thecase of multiplex analysis, the primer set for each target nucleic acidcan be equipped with a detectably different label, i.e. with a differentfluorescent spectrum.

In certain embodiments, the system can also comprise reagents for theamplification of the isolated DNA such as enzymes, DNTPs etc.

In certain embodiments, the system can also comprise a detection device.The detection device can be under the control of a control unit. Thedetection device is suitable for the detection of the amplified DNA orRNA and preferably for the detection of the labels that are incorporatedin the amplification products.

The detection device may detect based on label, length, mobility,nucleotide sequence, mass or a combination thereof. In certainembodiments a detection device can detect based on optical,electrochemical, magnetic or mobility (gel-electrophoresis). Inprinciple any suitable detection device known from prior art may beused.

In certain embodiments, the system also comprises a data collectiondevice to collect data obtained from the detection device.

In certain embodiments, the system also comprises a data processingdevice to process the data.

In one aspect of the present invention, there is provided a method forthe detection of the presence, absence and/or amount of a targetnucleotide sequence in a sample comprising one or more nucleic acidsequences, wherein the method comprises the steps of:

providing a sample from an organism;

performing steps for isolation of the nucleic acid sequences from thesample;

performing steps for amplification of (part of) the nucleic acidsequences to thereby provide amplicons;

detecting the presence, absence and/or amount of the ampliconscorresponding to the target nucleotide sequence amongst the nucleic acidsequences in the sample.

In certain embodiments, the method is performed in a cartridge asdefined in the present application.

In certain embodiments, the target nucleotide sequence can be selectedfrom the group consisting of DNA, genomic DNA, RNA, mRNA, cDNA,transgenic DNA, ETC.

In certain embodiments, the organism is a human, a non-human animal, amicro-organism or a plant.

In certain embodiments, the sample is tissue, bodily fluids such assputum, semen, blood, urine, and/or faces.

In certain embodiments, the target nucleotide sequence is an exogenoussequence.

In certain embodiments, the target nucleic sequence is a pathogen.

In certain embodiments, the sample comprising the nucleic acid sequencesis subjected to lysis to free the contained nucleic acid sequences. Incertain embodiments, the lysed sample is subjected to a sequence ofwashing and collecting steps as are themselves known in the art anddescribed in standard text books that aim at the isolation of thenucleic acids from the sample. These steps can be performed in a singlestep or as a sequence of multiple steps. After isolation of the nucleicacids from the sample, the nucleic acids can be subjected to anamplification reaction using primers that are selective for thedetection of the target nucleic acid.

Nucleic acid amplification methods usually employ two primers, dNTPs,and a (DNA) polymerase. A preferred method for amplification is PCR.“PCR” or “Polymerase Chain Reaction” is a rapid procedure for in vitroenzymatic amplification of a specific DNA segment. The DNA to beamplified is denatured by heating the sample. In the presence of DNApolymerase and excess deoxynucleotide triphosphates, oligonucleotidesthat hybridise specifically to the target sequence prime new DNAsynthesis. One round of synthesis results in new strands of determinatelength, which, like the parental strands, can hybridise to the primersupon denaturation and annealing. The second cycle of denaturation,annealing and synthesis produces two single-stranded products thattogether compose a discrete double-stranded product, exactly the lengthbetween the primer ends. This discrete product accumulates exponentiallywith each successive round of amplification. Over the course of about 20to 30 cycles, many million-fold amplification of the discrete fragmentcan be achieved. PCR protocols are well known in the art, and aredescribed in standard laboratory textbooks, e. g. Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1995).Other multiplex and/or isothermal amplification methods that may beapplied include e. g. LCR, self-sustained sequence replication (3SR),Q-β-replicase mediated RNA amplification, rolling circle amplification(RCA) or strand displacement amplification (SDA).

Detection of the labelled amplicons is performed by a detector to resultin detection data. The detector is of course dependent on the generalsystem with which the discrimination between the amplicons of the targetsequences is performed but is also depending on the label that ispresent on the primer, such as a fluorescent or a phosphorescent label.To discriminate between different target sequences in the samplepreferably a difference in fluorescence spectrum of the respectivecorresponding amplicons is used. In certain embodiments, at least one ofthe primers comprises a label, preferably the forward primer comprises alabel. The label can be selected from a large group, amongst otherscomprising fluorescent and/or phosphorescent moieties such as dyes,chromophores, or enzymes, antigens, heavy metals, magnetic probes,phosphorescent moieties, radioactive labels, chemiluminescent moietiesor electrochemical detecting moieties. In certain embodiments the labelis a fluorescent or phosphorescent dye. Examples of such dyes are FAM,HEX, TET, JOE, NED, and (ET-) ROX. Dyes such as FITC, Cy2, Texas Red,TAMPA, Alexa fluor 488™, BodipyFL, Rhodamine 123, R6G, Bodipy 530,Alexafluor™532.

By using different primer sets each containing a different label, thenumber of target sequences that can be discriminated in a sample andhence the number of target sequences in a sample that can be detectedcan be increased by using additional labels. The maximum number oflabels that can be used in one sample in a multiplex method is governedmostly by the limitations in the detection capabilities of the availabledetection platforms.

In certain embodiments, the amplification is performed using thePolymerase Chain Reaction with at least one forward and at least onereverse primer that are selective for the target sequence and not forany other sequence in the sample.

In certain embodiments, at least one of either the forward or thereverse primer is labelled.

In certain embodiments, the amplification step is preceded or replacedby an assay for the detection of nucleic acids in samples.

In certain embodiments, the amplicons are detected based on label,length, mobility, nucleotide sequence, mass or a combination thereof.

In certain embodiments, the amplicons are detected based on optical,electrochemical, or magnetic detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of a system according to an embodimentof the invention;

FIGS. 2-1 and 2-2 show a schematic block diagram of the architecture ofan embodiment of the system according to the invention;

FIG. 3 shows a schematic cross section (B-B in FIG. 4) of an embodimentof a cartridge according to the present invention; and

FIG. 4 shows a schematic top view/cross section (A-A in FIG. 3) of theembodiment of FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows an embodiment of a system for the detection of thepresence, absence and/or amount of a target nucleotide sequence in asample comprising one or more nucleic acid sequences, in generalindicated with the reference numeral 1. The system comprises a reusableapparatus 2 with a housing 3 (partly broken away).

In the apparatus 2 a recess 4 is provided. An exchangeable cartridge 5is removably positioned in this recess 4. The cartridge 5 may bereusable, recyclable or disposable.

In order to make detection possible the cartridge 5 comprisesintroduction means for the introduction of a sample, isolation means forthe isolation of DNA, amplification means for the amplification of DNA,and detection means for the detection of amplified DNA. The introductionmeans, isolation means, amplification means and/or detection means maybe arranged on the cartridge and/or in the reusable apparatus. Ingeneral it is preferred to arrange in the apparatus 2 all parts of thesystem 1 which normally do not come into contact with the sample. Thesample is held throughout the detection process in cartridge which worksas a cartridge.

Hereinafter a preferred embodiment of the arrangement of theintroduction means, isolation means, amplification means and/ordetection means is described. However, other embodiments are alsopossible.

The apparatus 2 comprises a control unit 7 for automatically controllingthe different steps of the detection process as will be describedhereinafter.

Further, the apparatus 2 comprises one or more actuation devices for theactuation of different elements arranged on the cartridge. Theseactuation devices may comprise one or more pump means actuation devicesfor the actuation of one or more pump means for pumping fluid, one ormore valve actuation devices for actuation of one or more valves beingarranged in a fluid channel in the cartridge, and other actuationdevices such as mechanical actuation devices for providing, for example,a rotary or translating movement to one or more parts of the cartridge.

In the apparatus a detection device is provided which may detect thepresence, absence and/or amount of DNA. For this purpose the DNA may beplaced in a detection chamber which is arranged on the cartridge. Thedetection device may work on an optical, electrochemical, or magneticprinciple as known from prior art. Any other suitable detection methodmay be applied.

The apparatus may further comprise a data collection device and a dataprocessing device to collect data obtained from the detection device andto process these data, respectively.

The apparatus 2 comprises a carrier 6 for supporting the cartridge 5.The carrier 6 is movable in a vertical direction between a lowerposition (in which the carrier is shown) and a higher position. In thelower position the cartridge 5 can be placed on or taken from thecarrier 6. The higher position is the working position in which thecartridge 5 is positioned during the detection process. In this higherposition the cartridge is clamped between the carrier 6 and the a numberof devices being arranged on the cartridge, such as pump means, valves,mechanical means, and a detection chamber may cooperate withcorresponding devices being arranged in the apparatus 2, such as pumpmeans, valve and other mechanical actuation devices, and a detectiondevice.

In an alternative embodiment it is also possible that a part of theapparatus 2 comprising the corresponding devices can be moved towardsand away from a cartridge placed in the apparatus 2.

In FIG. 2 a schematic block diagram is shown in which the differentprocess steps of the detection process using the method according thepresent invention are shown. This diagram is used to explain the mainarchitecture of the cartridge 5 and the relation between the apparatus 2and the cartridge 5.

In a first step (“sample insertion”) a sample is introduced in thecartridge 5. For this purpose the cartridge 5 comprises an introductiondevice with which a sample can be introduced in the cartridge 5. Theintroduction device may for example be any suitable device for theintroduction of a sample from a syringe or pipette or such, and maycomprise a holding or docking device, a one-way inlet valve, a septum,filters, and an overflow. After introduction of the sample this samplemay be guided to an introduction chamber.

In a second step (“lysis”) the sample is treated to provide any nucleicacids in the sample in a form that they can be isolated from the sample.This lysis step typically includes the lysis of the cells such that celland/or nuclear membranes are ruptured to thereby free the nucleic acidscontained therein. The lysis step is carried out in a lysis chamberwhich is part of a lysis device. This lysis chamber is in fluidcommunication with the introduction device for the sample, for instanceby means of a fluid channel. Pumping means may be provided for pumpingthe sample from the introduction chamber to the lysis chamber.

In a preferred embodiment the introduction chamber and lysis chamber arethe same chamber.

In an embodiment the lysis device comprises a physical or mechanicalmanipulation means for the lysis step. In another embodiment, or thesame embodiment, (also) chemical means can be used for lysis of thecells in the sample, such as a lysis buffer. Such lysis buffer may beheld before use in a separate lysis buffer container which is in fluidcommunication with the lysis chamber. A valve, preferably a one-wayvalve, may be provided in the fluid channel connecting the lysis buffercontainer and the lysis chamber.

Means for mixing can be provided to mix the sample and the lysis buffer.These mixing means may be actuated by the apparatus.

The lysis and possibly the mixing is carried out under control of thecontrol unit of the apparatus 2. The valves and pump means are actuatedby the valve and pump means actuation devices being arranged in theapparatus 2.

Any waste fluid that is produced by the lysis step can be discarded, forinstance to a waste device which may be present in the cartridge. Suchwaste device may be embodied as a waste chamber which is in fluidcommunication with the lysis chamber.

In a third step (“enrichment”), an enrichment device, being arranged inthe cartridge, enables the isolation of DNA from the lysed sample. Tothis end the enrichment device may be equipped with means for theisolation of DNA, such as magnetic particles.

The enrichment step is carried out in an enrichment chamber which is influid communication with the lysis chamber. In the fluid channel betweenlysis chamber and enrichment chamber a valve is provided to make itpossible that only a flow through the fluid channel is possible whenrequired. The valve may be actuatable by the valve actuation meansprovided in the apparatus.

In this embodiment, the DNA or RNA of the present invention is absorbedonto magnetic particles. The absorbed nucleic acid material can besubjected to one or more washing, draining and/or purifying steps toremove any unwanted material such as remains of biological materialcontained in the sample and other sample components that are not DNAand/or RNA. This washing and purifying step is shown as a fourth step“washing and purifying” in FIG. 2. However, the “washing and purifying”step can also be regarded as a part of the “enrichment” step. When theabsorbed DNA or RNA is of a desired purity, it can be desorbed or elutedfrom the magnetic particles. The washing and purifying step is carriedout in a washing chamber. In the present embodiment this washing chamberis the same as the enrichment chamber. However, in other embodiments aseparate chamber may be provided.

The cartridge 5 is provided with one or more washing buffer and elutionbuffer containers for holding the washing buffer(s) and elutionbuffer(s), respectively. Each of these washing buffer and elution buffercontainers is in fluid communication with the washing chamber, and againeach of the fluid channels providing this fluid communication isprovided with a valve, preferably a one-way valve. Similar containersmay be provided for any other reagents that are necessary for theenrichment step, i.e. the isolation of the DNA or RNA.

The valves of the enrichment device are actuated by the valve actuationdevice of the apparatus 2 and may be under control of the control unit7.

In an alternative embodiment the enrichment device can also be equippedwith physical or mechanical manipulation means of the fluids for mixing,separating and isolating the DNA or RNA. Such physical or mechanicalmanipulation means may be actuated by an actuation device of theapparatus 2 and may be under control of the control unit 7 of theapparatus.

Any waste produced from the enrichment step such as used buffers,washing fluids and the like can be guided to a waste device. This wastedevice which is part of the cartridge may be the same waste device asthe waste device described in the lysis device. As an alternative, thewaste devices of the lysis step and the enrichment step can be separatefor each different purpose or volume.

In a fifth step (“pre-amplification”) the total amount of DNA or RNA tobe analyzed may be increased by the use of a pre-amplification device.Subjecting DNA or RNA obtained from the isolation step to apre-amplification step can increase the total amount of DNA. This isadvantageously, especially in the case of multiplex analysis, wheremultiple tests are performed on the isolated DNA, for instance to detectthe presence absence or amount of multiple pathogens in one sample at atime.

The pre-amplification device comprises a pre-amplification chamber inwhich the pre-amplification is carried out. The pre-amplificationchamber may be the same chamber as or a different chamber than theenrichment chamber and/or washing chamber. The pre-amplification deviceis under the control of the control unit 7.

In the pre-amplification device, the isolated and purified DNA or RNAcan be pre-treated with, inter alia an pre-amplification buffer and incase of whole genome amplification, with enzymes and DNTPs. Before usethis pre-amplification buffer is held in a buffer container which is influid communication with the previous process chamber, for instance thewashing chamber. A valve may in the fluid channel providing the fluidcommunication.

The pre-amplification device can be connected to a waste device for thedisposal of materials.

In a sixth step (“amplification”) the isolated DNA, optionallypre-treated as described herein elsewhere, is subjected in theamplification device to an amplification treatment. The amplificationtreatment comprises bringing the isolated DNA in contact with a set ofPCR primers that are specific for the target nucleic acid, PCR enzymessuch as one or more polymerases and dNTPs.

For this purpose the amplification device comprises a plurality ofamplification chambers. The plurality of amplification chambers enablesthe isolated or pre-amplified DNA or RNA to be divided in portions anddistributed amongst the chambers. In each chamber, an amplification stepcan be performed using a different set of primers. In this manner,multiplex analysis is provided in that one sample can be analyzed forthe presence, absence or amount of different target nucleic acids. Inthe case of multiplex analysis, the primer set for each target nucleicacid can be equipped with a detectably different label, i.e. with adifferent fluorescent spectrum.

The cartridge may comprise reagents containers for holding reagents forthe amplification of the isolated DNA such as enzymes, DNTPs etc.

In a final step (“detection”) the amplified DNA or RNA and preferablythe labels that are incorporated in the amplification products aredetected. For this purpose the system 1 comprises a detection device.This detection device comprises a detection chamber which is arranged onthe cartridge 5. Other parts of the detection device may be arranged inthe reusable apparatus 2 as described herein above. The detectionchamber is in fluid communication with the one or more amplificationchambers for simultaneously or subsequently introducing the DNA or RNAout of the one or more amplification chambers. Valves may be provided inthe fluid channel connecting the detection chamber with the one or moreamplification chambers.

The detection device can be under the control of the control unit 7. Thedetection device may detect based on label, length, mobility, nucleotidesequence, mass or a combination thereof. In certain embodiments adetection device can detect based on optical, electrochemical, magneticor mobility (gel-electrophoresis). In principle any suitable detectiondevice known from prior art may be used.

The detected information may be collected by data collection means andprocessed by data processing means to come for instance to a certaindiagnose.

All fluid flows within the cartridge may be obtained by pump means whichare provided in the cartridge. Such pump means may work on the basis ofcompressing or expanding spaces in the cartridge, in particular thespaces of the respective process chambers, i.e. the introductionchamber, the lysis chamber, the pre-amplification chamber, the washingand purifying chamber, the amplification chamber the and detectionchamber, and the respective reagents containers. These pump means mayalso be of any other suitable type.

The pump means in the cartridge are actuated by the pump means actuationdevices provided in the apparatus 2. These pump means actuation devicesare under control of the control unit 7.

In the fluid paths or channels between the different process chambersi.e. the introduction chamber, the lysis chamber, the pre-amplificationchamber, the washing and purifying chamber, the amplification chamberthe and detection chamber, and the respective reagents containers,valves may be provided to only allow a flow when required. As most fluidwill pass the fluid channels only in one direction the valves arepreferably one-way valves.

The valves may be actuated by valve actuation devices which preferablyare arranged in the apparatus 2.

All steps as described above may be under control of the control unit 7.

FIGS. 3 and 4 show in more detail an embodiment of a cartridge generallyindicated with the reference numeral 10, in which the method asdescribed above can be performed. The cartridge comprises a generic part11 having a number of process chambers, and fluid handling systems aswill be described hereinafter.

The different parts of the cartridge 10 will hereinafter be described inthe order in which they will be used when a detection method fordetection of the presence, absence and/or amount of a target nucleotidesequence in a sample comprising one or more nucleic acid sequences isperformed.

The first application-specific part which is comprised in the cartridge10 is a pre-lysis device 12. This pre-lysis device 12 is configured toprocess a sample to a certain state which can be processed by thecartridge 10.

For example the sample may be provided in a solid state, for instancedried out blood, while the cartridge is designed to process a sample ina fluid state. In such case the sample has to be brought into a fluidstate before it can be processed in the cartridge. Such processing maybe performed by providing suitable enzymes in a suitable medium in thepre-lysis device 12. Such processes are known in the art, such as forexample trypsinization. By providing a pre-lysis device which can beconnected to the generic part 11, the processing of the sample to thedesired state can be performed without the need of transferring thesample after processing thereof therewith avoiding any chance oncontamination. The processing of the sample to the desired state may beperformed before or after that the pre-lysis device is connected to thegeneric part 11.

When no processing of the sample is needed, as the sample is already ina state which can be processed by the cartridge, the pre-lysis-devicemay also be indicated as a sample introduction device. The sampleintroduction device is then used to introduce the sample into thecartridge without risking any contamination, as the sample introductiondevice is designed to be connected to the generic part 11 for theintroduction of the sample in the cartridge 10.

When the sample is introduced in the cartridge 10, it may be pumped tothe lysis chamber 13. The generic part 11 of the cartridge 10 comprisesfluid handling means including pumps and valves for pumping the sampleto the different process chambers. In general the generic part 11comprises two main components 14, 15 which are placed against each otherwith interposition of a flexible membrane 16. The two main components14, 15 comprise recesses which together with the flexible membrane 16may form pump chambers, valves, fluid channels, fluid storage stationsand such.

In the cartridge shown in the drawings the sample will mainly be keptabove the flexible membrane, while pumps 17 and valves 18 are mainlyactuated from the bottom side of the flexible membrane 16. Fluid can bepumped in or out of a chamber by moving the flexible membrane toincrease or decrease the space within the chamber, respectively. Theflexible membrane can for example be moved by introducing air or fluidinto the space between the flexible membrane 16 and the component 15.The air or fluid may be introduced through the channels 19. The otherpump chambers may also be used as pump chambers in a corresponding way.Other means for moving the flexible membrane such as mechanicalactuators may also be used. The valves may be actuated by air or fluidpressure, mechanical actuation or any other suitable actuation device.The movement of the flexible membrane 16 with respect to the component14 may also be used to open and close a valve seat, whereby for examplein the closed position of a valve the flexible membrane 16 is heldagainst a channel end of the component 14.

In itself, such cartridge based systems having the type of pumps 17 andvalves 18 for the handling of fluids as described, have been disclosedbefore, however, but not for the purpose of the present invention.Reference is made, inter alia, to U.S. Pat. No. 6,156,270, USD37164, USD351913, U.S. Pat. No. 6,382,923, U.S. Pat. No. 6,663,359, U.S. Pat. No.6,416,293, U.S. Pat. No. 4,865,584 and U.S. Pat. No. 4,479,760.

In the lysis chamber 13 the sample will be lysed as here above describedin step 2 in relation to FIG. 2. A lysis storage 20 is provided to storea lysis buffer before it is pumped in the lysis chamber.

After the lysis step the sample may be pumped to a second processchamber 21 wherein the sample may be enriched in accordance with step 3and washed and purified in accordance with step 4 as described hereabove. Fluid storages 22 are provided for the storage of differentwashing and purifying buffers which may be used during the washing andpurifying steps. These fluid storages 22 are in fluid communication viavalves with the second process chamber 21.

After possible pre-amplification (as described in step 6 in relation toFIG. 2) which may also be performed in the second process chamber 21 orin the chamber 23, the sample may be introduced in the PCR body 24.

This PCR body 24 is a second application-specific part of the cartridge.The PCR body 24 is circular, disc shaped and connected with a click-fitconnection 25 to the generic part 11.

The PCR body 25 comprises six thermocycling chambers 26 so that six PCRprocesses can be simultaneously be performed on the sample. Such PCRamplification process is here above described as step 6 in relation toFIG. 2. Each of the thermocycling chambers 26 is provided with at leastone specific primer.

The PCR body 25 may be selected out of a group of different types of PCRbodies each comprising a different set of primers, a different number ofchambers and/or a different chamber size or geometry. For instance thePCR body comprising the primers can be selected on the basis of thepanels of bacteria/resistances that are to be detected, which selectionmay be specific for a particular assay or for a particular region, suchas Europe, Asia or Africa.

The primers are spotted on a wall of the thermocycling chambers, forinstance by an inkjet printing method, so that during storage of the PCRbodies no special measures have to be taken to avoid that the primersflow out of the PCR body, which would for instance be the case ifprimers in a fluid state would be used. In such case a seal or separatesealed chamber may be provided for holding the primers any otherapplication-specific fluid before use thereof.

After the amplification step the amplified DNA or RNA and preferably thelabels that are incorporated in the amplification products are pumped tothe detection device 27. This detection device or at least a partthereof is a third application-specific part of the cartridge 10, whichis a separate part and can be connected to the generic part 11. In theshown embodiment the detection device is connected to the generic part11 by a click-fit connection.

Depending on the type of detection method and/or detection means (asdescribed in this application; in particular step six described inrelation to FIG. 2), a detection device may be chosen out a series ofdifferent application-specific detection devices which may be speciallydesigned for each respective detection method. In some cases the type ofdetection device that will be used in the cartridge 11 will be dependenton the type of PCR body which is used for the amplification process.Then the choice of a PCR body will automatically lead to a choice of thedetection device.

The generic part 11 and the application-specific parts are provided withan identification device, so that after assembly of the generic andapplication-specific parts it can be checked whether the correctcombination is made. Possibly, a more advanced identification system isused, as for instance a RF-tag, which comprises identification tagswhich automatically can be checked and of which possibly even thehistory can be tracked. Such checking and history tracking can becontrolled by the control unit of the reusable apparatus as a step inthe procedure for processing the sample in the cartridge.

An additional advantage of the construction of the present cartridgewith a generic part and one or more application-specific parts is thatthe connection between the generic part and each of theapplication-specific parts can be easily made airtight, so that theentire space wherein the sample and other fluids used in the cartridgemay be closed from the environment In this way contamination of thesample during introduction of the sample in the cartridge and processingthereof is avoided and, since the sample is in a closed environmenthaving its own internal pressure, the processing of the sample can beperformed independent of the air pressure in the direct environment, andalso independent of other environmental conditions as humidity. Thismakes a more reliable processing of the sample possible.

It is contemplated that the cartridge according to the present inventionmay comprise other application-specific parts than theapplication-specific parts identified in the above description. Theapplication of such other separate application-specific parts in thecartridge are deemed to fall within the scope of the present invention.Examples of such application-specific parts may comprise fluidcontainers which contain a fluid such as enzymes, reagents, and otherchemical substances for a specific application, mixing devices and othermechanical manipulation devices with different geometries or sizes for aspecific application and others.

The invention may also be used for specific parts of the cartridge whichhave to be pre-treated or have to be kept at a certain temperature whichis not desired or required for the other parts of the cartridge. Forinstance, the provision of a separate fluid container which can be usedin the pre-treatment or stored at a different location, and which canconsequently be connected to the generic part of the cartridge beforeuse, may be very useful since the risk on contamination of that part, inparticular the fluid therein is avoided, since the fluid does not haveto be transferred from a container to the cartridge in an openenvironment.

Such use of a separate part is regarded to be application-specificwithin the meaning of the present invention, even if the same part isused in a number of different applications. An example of such separatepart is a separate fluid container for a so-called PCR master mix whichhas to be stored at a low temperature before use on the cartridge. Justbefore the cartridge is introduced into the reusable apparatus, theseparate fluid container is connected to the generic part of thecartridge, for example by a click fit connection.

What is claimed:
 1. A cartridge for the detection of the presence,absence and/or amount of a target nucleotide sequence in a samplecomprising one or more nucleic acid sequences, characterized in that thecartridge comprises a generic part and one or more separateapplication-specific parts, which are connectable to the generic part.2. A cartridge according to claim 1, wherein one of the one or morespecific parts is a PCR body having one or more thermocycling chambersand comprising a number of primers.
 3. A cartridge according to claim 2,wherein at least one primer is arranged in each one of the one or morethermocycling chambers.
 4. A cartridge according to claim 2, wherein atleast one of the number of primers is spotted on the PCR body.
 5. Acartridge according to claim 2, wherein the PCR body is disc-shaped. 6.A cartridge according to claim 2, wherein the PCR body comprises one ormore thermal masses.
 7. A cartridge according to claim 1, wherein one ofthe one or more application-specific parts is a detection device.
 8. Acartridge according to claim 2, wherein the detection device is selectedon the basis of the primers in the PCR body.
 9. A cartridge according toclaim 1, wherein one of the one or more application-specific parts is asample introduction device configured to prepare a sample to a specificstate.
 10. A cartridge according to claim 9, wherein the sampleintroduction device is a pre-lysis device configured to prepare a sampleto a specific state.
 11. A cartridge according to claim 1, wherein atleast one of the one or more application-specific parts is connectableon the main body with a click-fit connection.
 12. A cartridge accordingto claim 1, wherein each space for holding the sample or part thereof inthe generic part and the one or more application-specific parts isair-tight.
 13. A cartridge according to claim 1, wherein at least one ofthe one or more of the application-specific parts is provided with anidentification device.
 14. A cartridge according to claim 12, whereineach of the one or more application-specific parts and the generic partis provided with an identification device.
 15. System for the detectionof the presence, absence and/or amount of a target nucleotide sequencein a sample comprising one or more nucleic acid sequences, said systemcomprising a reusable apparatus, wherein said apparatus is configured toreceive a cartridge according to claim 1 and to control a process forthe detection of the presence, absence and/or amount of a targetnucleotide sequence in the sample, being present in said cartridge. 16.A method for the detection of the presence, absence and/or amount of atarget nucleotide sequence in a sample comprising one or more nucleicacid sequences, wherein the method comprises the steps of: providing asample from an organism; performing steps for isolation of the nucleicacid sequences from the sample; performing steps for amplification of(part of) the nucleic acid sequences to thereby provide amplicons;detecting the presence, absence and/or amount of the ampliconscorresponding to the target nucleotide sequence amongst the nucleic acidsequences in the sample, characterized in that the method is beingperformed in a cartridge according to claim
 1. 17. A method according toclaim 1, wherein the method further comprises the selection of one ormore application-specific part and mounting said application-specificpart on the generic part of the cartridge.